Methods and Results To test the hypothesis that long-term etomoxir treatment improves the performance of hypertrophied ventricle, sham-operated rats and rats with ascending aorta constriction were treated with racemic etomoxir (15 mg/kg per day) for 12 weeks. Left ventricular geometry, dynamics of isovolumic contractions, as well as myosin isozymes as marker of etomoxir-induced phenotype changes were assessed. Etomoxir stimulated (P<.05) slight hypertrophic growth in right and left ventricles of sham-operated rats as well as in right ventricles but not in overloaded left ventricles of rats with aortic constriction. In all treated rats, etomoxir increased (P<.05) maximal developed pressure, left ventricular pressure-volume area, and ±dP/dtmax. Enhanced values (P<.05) of derived indexes of myocardial performance (normalized stress-length area, maximal rate of wall stress rise, and decline) indicated that myocardial changes were responsible for the improved performance. The etomoxir treatment increased selectively (P<.05) the proportion of myosin V1 in pressure-overloaded left ventricles.

Conclusions The long-term treatment with etomoxir improved functional capacity of pressure-overloaded left ventricle, which can be attributed to an enhanced myocardial performance. Chronic carnitine palmitoyltransferase-1 inhibition may thus represent a candidate approach for developing novel agents that are useful in the prevention of undesirable consequences of pressure overload–induced cardiac hypertrophy.

Current drug treatments of the failing heart syndrome are targeted primarily at the peripheral consequences of pump failure or modulate acutely ion homeostasis of the heart muscle cell. Since various subcellular organelles are altered in hypertrophied and failing heart, the question arises whether the poor life expectancy of patients with left ventricular dysfunction could be improved by preventing changes in molecular structures of overloaded cardiomyocytes.1 However, it remains undefined which features of hypertrophied myocardium should be modified to prevent development of heart failure and what type of pharmacological intervention could be useful.

The isoform switch in myosin heavy chain (MHC) expression during the development of heart hypertrophy and failure was the first identified molecular change in overloaded myocardium and the first example of the reactivation of genes that are normally expressed during embryonic development.23 Preferential β-MHC synthesis is, however, only one feature of the myocardial hypertrophic response, which involves expression of other embryonic markers as well as quantitative and qualitative changes in expression of genes encoding various contractile and noncontractile proteins.4 Some changes in gene expression are transient, for example, induction of expression of immediate early genes,4 whereas others such as the decrease in sarcoplasmic reticulum (SR) Ca2+-ATPase expression are detected only at later stages.5 β-MHC mRNA begins to accumulate with the onset of overload-induced hypertrophy and remains elevated as long as the load persists.3 Activation of the β-MHC gene and a deactivation α-MHC gene could thus be considered a hallmark of phenotype alterations in overloaded rat heart. The isoform switch in MHC expression occurs in all cardiac tissues and species tested.6 However, the capacity to increase the β-MHC proportion depends on the initial phenotype. This capacity is high in rat ventricles and in human atria, which express preferentially α-MHC but is low in human ventricles with mainly β-MHC.

Functional consequences of different myosin phenotypes are still not entirely understood. The change from α-MHC to β-MHC results in a lower myosin ATPase activity and reduced shortening velocity.7 This isoform shift, by slowing cross-bridge cycling, reduces myocardial contractility. However, the preferential synthesis of β-MHC improves the mechanical efficiency of the overloaded heart.8 Thus although expression of β-MHC gene has a negative inotropic effect, it is also energy sparing.9

The well-documented potential of thyroid hormone to increase myocardial contractility and α-MHC synthesis10 resulted in the successful approach of improving the function of infarcted rat ventricle with l-thyroxin11 as well as thyroid hormone analogues that are deficient in positive chronotropic effects.12 Recently, it has been shown that etomoxir, a carnitine palmitoyltransferase-1 (CPT-1) inhibitor, influences myosin isoenzyme distribution and expression of SR Ca2+-ATPase similar to thyroid hormone. In rats, long-term treatment with etomoxir induced cardiac hypertrophy, redistributed the myosin isozyme population from V3 to V1, and increased SR Ca2+ pump activity.1314151617 However, it remained unresolved whether the shift in myosin isozymes was associated with an altered performance of pressure-overloaded rat ventricles.

To address this question, rats with constriction of the ascending aorta were treated with etomoxir. Left ventricular performance of isovolumically beating hypertrophied hearts was assessed 12 weeks after inducing hemodynamic overload as well as the etomoxir treatment. The approach permits a load-independent evaluation and comparison of left ventricular and myocardial function despite alterations that occur in cardiac mass, geometry, and myocardial contractility.18 To examine whether changes in myocardial performance could be accounted for by an altered MHC expression, the myosin isozyme population was assessed at the protein level. The study demonstrates that long-term treatment with etomoxir improves the functional capacity of pressure-overloaded rat left ventricle that was attributed to an enhanced myocardial performance. Chronic CPT-1 inhibition may thus represent a candidate approach for developing novel agents that are useful in the prevention of undesirable consequences of pressure overload– induced cardiac hypertrophy.

Methods

Animal Model

Male 3-week-old Wistar/WU rats were purchased from Charles-River (Kissleg, Germany) and housed at 21° to 23°C on 12:12-hour light-dark cycle. The rats had free access to tap water and regular chow (Ssniff of Plange). Handling of animals and all experimental procedures were in accordance with the Institutional Guidelines of the University of Tübingen (Germany).

The ascending aorta was banded with a 3-0 silk suture ligature tied against an 0.8-mm blunt steel wire at a body weight of 90 to 100 g under Hypnorm (fluanisone/fentanyl-dihydrogencitrate) anesthesia (1 ml/kg IP). The wire was removed, whereby the aorta was constricted to 60% to 70% of the original diameter. After 12 weeks, the mean left ventricular aortic pressure gradient was 87±4 mm Hg, with no significant difference between etomoxir-treated and nontreated animals. Ascending aortic constriction was performed in 28 rats, 14 rats remained untreated, and 14 rats were treated daily with 15 mg/kg body wt racemic etomoxir starting 1 day after surgery. Age-matched control animals underwent a right thoracotomy, and the ascending aorta was isolated but not constricted. Starting 1 day after surgery, 14 sham-operated rats received 15 mg/kg per day racemic etomoxir and 14 sham-operated rats remained without treatment. Etomoxir was generously provided by Dr H.P.O. Wolf of Byk Gulden, Konstanz, Germany, and was given in the drinking water. The dose was maintained by monitoring the daily water consumption and body weight.

Measurement of Left Ventricular Isovolumic Contraction

Animals were studied 12 weeks after surgery. The measurements were performed in open-chest rats under urethane anesthesia (1.2 g/kg body wt IP) as described previously.1819 A mediosternal thoracotomy was performed and the left ventricle was pierced at the apex with a steel cannula No. 1 connected to a Gould-Statham P23XL pressure transducer (Gould Electronics). The right carotid artery was cannulated with a polyethylene tubing (0.5-mm inner diameter) and forwarded to the aortic arch. The tubing was connected to a second Gould-Statham P23XL pressure transducer. Left ventricular pressure, left ventricular diastolic pressure (high amplification of left ventricular pressure), time derivative of ventricular pressure (dP/dt), and aortic pressure were recorded simultaneously on a Hellige Recomed recording system.

For monitoring isovolumic contractions, the ascending aorta was clamped above the aortic valve for 6 to 8 seconds with a forceps, as verified by the absence of pulsatile pressure in the aortic arch. Small end-diastolic volumes, that is, low preload values, were achieved by a short tightening of a string around the inferior vena cava. The preload increased gradually after relieving the inferior vena cava flow during clamping of the aorta. This procedure was repeated four to six times. The functional analysis was based on the recording exhibiting the highest systolic pressures development.

Left ventricular passive pressure-volume relations were assessed after recording isovolumic contractions. The atrioventricular groove was ligated with a silk string, and the right ventricle was emptied by incision. The left ventricle was filled with a defined volume of saline and emptied in 50-μL steps while recording the passive left ventricular pressure. Three reproducible pressure-volume curves were generated within 3 to 4 minutes after the ligation. No effects of anoxia on the pressure-volume relation could be detected within this period of time. Using the passive pressure-volume relation, end-diastolic cavity volumes required for further analysis were derived from the measured end-diastolic pressures.

Data Analysis

The approach permitted the construction of complete left ventricular pressure-volume and stress-length diagrams. Left ventricular and myocardial function were thus assessed independent of left ventricular mass, geometry, and loading conditions.18 Systolic peak pressures that are equivalent to end-systolic pressures under the isovolumic conditions were plotted against end-diastolic volumes resulting in end-systolic pressure-volume curves. All auxotonic pressure-volume values had to reside within this isovolumically determined end-systolic pressure-volume relation. Thus the area between end-systolic and end-diastolic pressure-volume curves up to maximum end-systolic pressure was used as an index of left ventricular working capacity. Transformation of the left ventricular pressure-volume diagram to the stress-length relation permitted the evaluation of myocardial performance when left ventricular mass and geometry are altered. Myocardial contractility was evaluated on the basis of normalized stress-length area, that is, the area between the end-systolic and end-diastolic mean wall stress versus normalized midwall circumference (length) curves. In analogy with papillary muscle, the normalized midwall circumference was calculated as the ratio between a given midwall circumference and the midwall circumference associated with peak developed wall stress.18 Pressure-volume data were transformed into stress-length data using a thick-walled spherical shell. Since calculations assumed that the specific density of myocardium was 1 g/cm3, left ventricular weight in grams equaled the ventricular wall volume in cm3.

Mean (systolic or diastolic) wall stress (ς) was derived from the following formula20 : ς=P/{[(V+W)/V]2/3−1}, where P is left intraventricular pressure, V is left ventricular cavity volume, W is left ventricular wall volume. Since the contraction was isovolumic, end-diastolic volume derived from the passive pressure-volume curve was identical with the left ventricular cavity volume (V) during the respective beat. Midwall circumference (CR) was calculated according to CR=π{(3/4π)1/3[(V1/3+(V+W)1/3]}. To evaluate the velocity of contraction and relaxation at the myocardial level, rate of mean wall stress rise (+dς/dt) or decline (−dς/dt) was calculated using the recorded ±dP/dt values by ±dς/dt=±(dP/dt)/{[(V+W)/V]2/3−1}.

Myosin Isozymes

The proportion of myosin isozymes was determined by nondissociating polyacrylamide gel electrophoresis in the presence of pyrophosphate.13 Portions (30 to 50 mg) of the left ventricle frozen in liquid nitrogen were used. The myosin isozymes were stained with Coomassie brilliant blue R250 and the gels were scanned using a Quick Scan densitometer (Helena Laboratories). The isozymes V1, V2, V3 were quantitated by measuring peak heights. α-MHC was calculated using the equation

Statistical Analysis

Normality of distribution was checked by the Kolmogoroff-Smirnoff test and equality of variances according to Cochran. Multiple comparisons were made by one-way and two-way ANOVA and the post hoc Newman-Keuls test (Statistica/w, Statsoft). Statistical significance was assumed at P<.05.

Results

Treatment with racemic etomoxir (15 mg/kg per day) induced slight hypertrophy of both left and right ventricles in sham-operated rats as well as right ventricles of rats with aortic constriction (Table 1⇓). The aortic constriction resulted in a concentric remodelling of the left ventricle (Table 1⇓). The main effect (two-way ANOVA) of aortic constriction and of etomoxir treatment was to increase developed pressures, left ventricular pressure-volume areas, as well as positive and negative dP/dtmax (Figs 1⇓ and 2⇓ and Table 1⇓). There were no significant interactions, that is, the effects were additive, between pressure overload and etomoxir treatment for these parameters, except for −dP/dtmax. The etomoxir-induced increase in −dP/dtmax values was significantly more pronounced in rats with aortic constriction than in sham-operated rats. When compared with untreated rats with pressure overload, etomoxir-treated rats with aortic constriction exhibited a significant improvement in developed pressures (Fig 1A⇓), left ventricular pressure-volume areas (Table 1⇓), as well as ±dP/dtmax values (Fig 2A⇓ and B).

Body Weight, Right and Left Ventricular Weights, Left Ventricular Passive Diastolic Dimensions, Left Ventricular Pressure-Volume Area, and Normalized Stress-Length Area

An increase in left ventricular pressure-volume areas and ±dP/dtmax values of untreated rats with aortic constriction reflected the enhanced performance of ventricles with a greater muscle mass. However, derived indices of myocardial performance, that is, normalized stress-length area (Table 1⇑ and Fig 1B⇑) and maximal rates of wall stress rise and decline (Fig 2⇑, C and D), were reduced. By contrast, the etomoxir treatment increased these parameters. A significant interaction (two-way ANOVA) between pressure overload and etomoxir treatment was observed for −dς/dtmax but not +dς/dtmax or stress-length area. As a consequence, the etomoxir treatment improved −dς/dtmax more markedly than +dς/dtmax or stress-length area.

The left ventricular pressure overload decreased the proportion of myosin V1 and correspondingly increased the proportion of myosin V3 (Table 2⇓). The etomoxir treatment had an opposite effect: it increased the myosin V1 proportion and decreased the myosin V3 proportion (Table 2⇓). A significant interaction (two-way ANOVA) between pressure overload and etomoxir treatment was observed for the myosin isozymes proportion, indicating that etomoxir affected specifically myosin alterations induced by left ventricular pressure overload.

The percentage of α-MHC correlated significantly with derived parameters of myocardial performance, that is, stress-length area as well as rates of wall stress rise and decline (Fig 3⇓, B, D, and F). The dependence of ventricular function on both ventricular mass and myosin isozymes became apparent when relationships between parameters of ventricular function and the proportion of α-MHC were assessed. No significant correlation was observed when all groups were combined. By contrast, when groups with comparable ventricular weight, that is, sham-operated or pressure-overloaded rats, were analyzed, the proportion of α-MHC was correlated (P<.001) both with pressure-volume area and ±dP/dtmax. Two separate regression lines could be identified. One regression line for sham-operated rats and one for rats with aortic constriction (Fig 3⇓, A, C, and E). When the effect of ventricular mass was taken into account by transforming the pressure-volume area into stress-length area and the ±dP/dtmax values into ±dς/dtmax values, the two separate regression lines merged into one (Fig 3⇓, B, D, and F).

Discussion

The increase in the pressure-volume area and higher rates of pressure rise and decline of untreated pressure-overloaded left ventricles are signs of improved pump performance. A more thorough analysis demonstrates, however, specific defects at the myocardial level. To account for effects of ventricular mass and chamber volume on ventricular pump performance and to assess myocardial function, developed pressure was normalized by calculating mean wall stress and end-diastolic volume by calculating normalized midwall circumference. The transformation of the pressure-volume area and ±dP/dtmax values into stress-length area and ±dς/dtmax values revealed a depressed myocardial function of overloaded left ventricles (Figs 1⇑ and 2⇑ and Table 1⇑).

The two different correlation lines between indices of ventricular function and α-MHC proportion for the hypertrophied and nonhypertrophied ventricles also demonstrate that pump function is affected both by ventricular as well as myocardial parameters. Although the indices of ventricular function were increased in overloaded left ventricles despite decreased α-MHC proportion, the values were lower than those that would be extrapolated for hypertrophied ventricles with an extrapolated α-MHC proportion of 60% to 80%. These values were partially reached after etomoxir treatment (Fig 3⇑, A, C, and E).

Etomoxir improved left ventricular as well as myocardial performance without inducing a significant additional left ventricular hypertrophy. It is therefore plausible that the etomoxir treatment partially reversed the derangement underlying the decreased myocardial performance. By contrast, the modest enhancement of stress-length area and ±dς/dtmax values of sham-operated etomoxir-treated rats indicates that the observed improved left ventricular performance was only partially due to an enhanced myocardial performance and that the increased left ventricular mass had a contributing effect.

The mechanisms by which the etomoxir improved myocardial performance are not fully understood. Inhibitors of CPT-1 have no immediate effects on cardiac performance in control animals.21 It is therefore unlikely that the improvement of left ventricular function observed in this study is a consequences of acute metabolic alterations caused by CPT-1 inhibition. There is, however, increasing evidence that etomoxir improves left ventricular performance by phenotypic modification of hypertrophied ventricles. A chronic CPT-1 inhibition induced not only moderate cardiac hypertrophy but also increased α-MHC expression.1314151617 The effect of etomoxir on cardiac isomyosins and SR were more pronounced in functional states characterized by a reduced isomyosin V1 proportion and related SR parameters.1517 This is confirmed in the present study, in which etomoxir treatment is shown to increase significantly the proportion of isomyosin V1 of overloaded hearts but not of control hearts (Table 2⇑). Limited effect of etomoxir on V1 proportion in sham-operated rats may be related to the high initial percentage of V1 isomyosin in these young animals. A coordinated action on both myosin isoenzymes and SR maybe responsible for the modest enhancement of myocardial performance in etomoxir treated sham-operated rats.15

The significant correlation between the indices of left ventricular or myocardial performance and the percentage of α-MHC (Fig 3⇑) demonstrates an important influence of MHC on left ventricular performance. The question arises whether the observed correlation is causal or not. A number of studies have suggested that ventricular and myocardial function may be related to myosin isozyme composition.102223 A higher proportion of α-MHC is expected to increase the rate of cross-bridge cycling, and one could infer an increased rate of pressure rise7 but also increased rate of pressure decline.24 Enhanced developed pressures are typically related to a greater availability of activator calcium and faster relaxation to an increased rate of activator calcium removal.25 In the present study, the α-MHC proportion was correlated with indices that characterize both contraction and relaxation (Fig 3⇑). It thus appears that the changes in the α-MHC percentage after etomoxir treatment might also be associated with alterations in the activity of organelles responsible for the activator calcium cycling.

Similar to etomoxir, enhanced thyroid influences increased the proportion of myosin V1 that was associated with positive inotropic and lusitropic effects.10 Vice versa, in hypothyroidism, in which β-MHC predominates, contraction and relaxation were slowed.710 It should be pointed out that the present experiments were not designed to identify molecular processes involved in the improved function of overloaded hearts arising from etomoxir treatment. The study nonetheless supports the contention that the partial prevention of a decrease in α-MHC accounts for the improvement of left ventricular performance.

In the present approach, evaluation of isovolumic contractions was preferred to distinguish myocardial and ventricular determinants of pump function of hypertrophied left ventricles. It was thus not possible to assess the influence of etomoxir treatment on left ventricular ejection. Other limitations relate to nonsimultaneous pressure and volume measurements, exclusion of right ventricular influences, assumptions on left ventricular geometry, and physical properties of the myocardium required for converting pressure-volume data to stress-strain data. Despite these limitations, the present comparative study clearly demonstrates that treatment with etomoxir improves myocardial function in rats with chronic left ventricular pressure overload. Since additional hypertrophy was absent in pressure-overloaded left ventricles after etomoxir treatment, this novel conclusion can be derived from directly measured values, ie, developed pressure and ±dP/dtmax, which do not involve transformations. A noteworthy finding was that the small dose of etomoxir used (15 mg/kg per day racemic form equivalent to 7.5 mg/kg per day of biologically active enantiomeric (+)-etomoxir) induced an improvement of left ventricular function that occurred independent of hypertrophy. In accordance, it was previously shown that 5 mg/kg per day enantiomeric (+)-etomoxir increased rate of SR Ca2+ uptake but not left ventricular weight,13 whereas higher doses resulted in cardiac hypertrophy.14151617 Further work is required to assess to what extent the present etomoxir treatment interfered with endogenous changes in fuel metabolism of hypertrophied hearts.262728

Indices of ventricular function depend also on heart rate and ventricular load. It is, however, unlikely that these variables significantly influenced the present results. The overall frequency of isovolumic contractions was 329±22 (bpm), with no significant differences within experimental groups. Furthermore, the differences in functional parameters were detected over the whole range of physiologically encountered preload values. Since the evaluated contractions were isovolumic (with the exception of coronary flow), the afterload was maximal for a given preload.

Acknowledgments

The study was supported by the Deutsche Forschungsgemeinschaft (Ja 172/14-1, Ru 245/6-2), the Alfred-Teufel- Stiftung, and the Slovak Grant Agency No. 1/4114/97 and 1/4133/97. The experimental part of this study was performed at the Institute of Physiology II, University of Tübingen, Germany.